CN1995380B - Method for detecting and identifying mycobacterium tuberculosis strain and its dedicated reagent kit - Google Patents
Method for detecting and identifying mycobacterium tuberculosis strain and its dedicated reagent kit Download PDFInfo
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- CN1995380B CN1995380B CN200610000496A CN200610000496A CN1995380B CN 1995380 B CN1995380 B CN 1995380B CN 200610000496 A CN200610000496 A CN 200610000496A CN 200610000496 A CN200610000496 A CN 200610000496A CN 1995380 B CN1995380 B CN 1995380B
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Abstract
The invention discloses a testing and identifying method of mycobacterium and specific agent box, which consists of atopic mycobacterium primer and mycobacterium testing chip, wherein the chip contains mycobacterium testing probe, which is composed of atopic probe, composite group atopic probe and 27 atopic probes. The invention utilizes the agent box to detect mycobacterium based on genome DNA of mycobacterium as mould, which proceeds PCR augmentation to obtain PCR augmentation product to cross the chip to judge the mycobacterium according to crossing signal.
Description
Technical field
The present invention relates to the method and the dedicated kit thereof of a kind of detection and identification of mycobacterium bacterial classification.
Background technology
In microorganism classification, mycobacterium belongs to Prokaryota, Firmicutes, Schizomycetes, actinomycetales, mycobacteriaceae, Mycobacterium.Bacterial classification is various in the Mycobacterium, and the mycobacterium that Bergey ' s manual of systemativebacteridogy (the outstanding systematic bacteriology handbook of uncle) is confirmed has 54 kinds.Except that mycobacterium tuberculosis complex bacterial classification (mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum and mycobacterium microti) and Mycobacterium leprae, other bacterial classification is referred to as non-tuberculous mycobacteria (NTM) in the Mycobacterium.Kind surplus human disease's NTM had 20, the mycobacterium that wherein grows slowly comprises that mycobacterium kansasii, Mycobacterium marinum, ape and monkey mycobacterium, Mycobacterium scrofulaceum, Soviet Union add branch bacillus, mycobacterium avium, Mycobacterium intracellulare, mycobacterium xenopi, mycobacterium buruli, Ma Ermo mycobacterium, Amur mycobacterium, it has been generally acknowledged that the also visible case report of mycobacterium gordonae, achromatic mycobacterium, soil mycobacterium, mycobacterium triviale, Asia mycobacterium of non-virulent in addition in the past; Fast growth mycobacterium comprises mycobacterium fortuitum, Mycobacterium chelonei, mycobacterium abscessus, even the metatroph of thinking in the past, not following several 10 examples of case have now been reported as smegmatis mycobacterium, and idol has the AIDS disseminated infections, and little yellow mycobacterium also can cause the lung and the infection of joint once in a while.In recent years, tuberculosis and the sick sickness rate of non-tuberculous mycobacteria that is worldwide caused by mycobacterium tuberculosis (MTB) and NTM obviously raises.
At present, tuberculosis clinical labororatory mycobacterium bacteriological detection, authentication method have:
1, smear staining: microscopy is seen acid-fast bacilli, the report acid-fast stain positive (+～++ ++), can not determine that the antiacid mycobacterium of finding is MTB or NTM, need to identify and can determine through mycobacteria strain.
2, separation and Culture: adopt solid mediums such as improvement Luo Shi, see colony growth, the report mycobacterium cultivation positive (+～++ ++), adopt Bactec-TB960 culture systems or Bact ALERT 3D culture systems, after instrument is judged to be the positive, get the acid-fast stain of culture smear, positive person reports that the mycobacterium cultivation is positive.Mycobacterium is cultivated the positive and can not determine that it is MTB or NTM, needs to identify and can determine through mycobacteria strain.
3, mycobacteria strain is identified
(1), MTB and NTM differentiate: mainly according to growth test on the PNB substratum, (-) is judged to be MTB, and (+) is judged to be NTM.
(2), mycobacteria strain identifies: according to growth characteristics, nearly 20 test index analysis-by-synthesis result of determination such as growth test and multiple biochemical reaction on the multiple differential medium.
This method for identification of mycobacterium species has following shortcoming: a, authentication method is numerous and diverse, need carry out nearly 20 tests.B, the required bacterial cultures amount of qualification test are bigger, and the cultivation of need going down to posterity after obtaining just for the separation and Culture thing is usually tested with subculture.Just for separation and Culture thing → subculture → qualification test → report the result, the mycobacterium that grows fast needed for 3～4 weeks, and the mycobacterium that slowly grows needed for 8～12 weeks.C, result precision are poor: between same bacterial classification different strains, the outstanding systematic bacteriology handbook of uncle is reported about 15% biology phenotype and is existed heterogeneously, makes bacterial classification be difficult to clearly identify or identify mistake.
The commercial kit that is used for mycobacterium gene test and evaluation mainly is divided into following two classes:
1, be used for that mycobacterium tuberculosis complex detects and the test kit identified (Li Guoli, the theory and practice of tuberculosis molecular Biological Detection method are seen: Xiao's peace chief editor. the tuberculosis prevention and treatment new development. Shanghai: press of Fudan University, 2004.128-135; Ministry of Health Clinical Laboratory center. appendix 3: relevant PCR reagent, instrument producer reagent, instrument explanation. the clinical gene amplification diagnostic test chamber data compilation 104-106 of Technician Training class, 109-111,118-120)
(1), the Amplicor test kit (Amplicor mycobacteriumtuberculosis) produced of German Roche company: design Mycobacterium primer amplified mycobacterium for target gene with 16S rRNA gene (16S rDNA), with the inner mycobacterium tuberculosis complex distinguished sequence design of amplified fragments hybridization probe, adopt PCR-micropore plate hybridization method.
(2), domestic many companies produce fluorescent PCR kit: the IS6110 special with mycobacterium tuberculosis complex designs primer and probe for target-gene sequence, adopts Taqman technology and molecular beacon (molecular beacon) technology respectively.
Above-mentioned two class test kits are used for detecting identifies the clinical sample mycobacterium tuberculosis complex, can not detect, identify NTM.Fluorescent PCR needs the fluorescent PCR instrument, and price is more expensive.
2, be used for test kit (the Tortoli E that mycobacteria strain is identified, Nanetti A, Piersimoni C, et al.Performance assessment of new multiplex probe assay for identification ofmycobacteria.J clin Microbiol, 2001,39:1079-1084; Miller N, Infante S and ClearyT.Evaluation of the LipA MYCOBACTERIA assay for identification of mycobacterialspecies from BACTEC 12 B bottles.J Clin Microbiol, 2000,38:1415-1919)
(1), the Accuprobe Mycobaterium test kit of U.S. gen-probe company production: with 16S rRNA is target sequence; design mycobacterium tuberculosis complex, the compound group of mycobacterium avium, mycobacterium avium, Mycobacterium intracellulare, mycobacterium kansasii and 6 kinds of acridinium ester (acridinium ester of mycobacterium gordonae; AE) the cDNA probe of mark; employing solution hybridization technology-hybridization protection test (Hybridization protect assay, HPA).The ultimate principle of HPA and method are: AE is a kind of chemiluminescent substance, and the group of AE mark or specific specificity probe are hybridized with the rRNA of mycobacterium release to be measured after treatment in liquid phase; Reagent brings Selection In; 1,000,000 times have been differed with the probe of target complement sequence hybridization with free probe hydrolysis rate under the selective reagents effect of not hybridizing; the rapid hydrolysis of free probe destroys; lose chemiluminescent properties; and the probe after the hybridization is protected; be not hydrolyzed destruction within a certain period of time, produce chemoluminescence, detect its relative light unit result of determination by chemiluminescence detector.
The advantage of this test kit is that method is easy, quick, reports the result in 2 hours.Its limitation is only to be applicable to the mycobacterium culture is identified; Test kit only comprises 6 kinds of probes, the compound group of mycobacterium tuberculosis complex, mycobacterium avium, mycobacterium avium, Mycobacterium intracellulare, mycobacterium kansasii and mycobacterium gordonae bacterial classification that can only the included probe of identification kit, other pathogenic mycobacteria strains of clinical visible must be identified by additive method; Test need chemiluminescence detector and test kit price are more expensive.
(2), LipA (Line Probe assay) the Mycobacteria test kit that Belgium Innogenetics company produces: with pcr amplification and reverse making nucleic acid molecular hybridization is principle, adopt 16S-23S rDNA internal transcribed spacer district (ITS) to design 14 mycobacterium oligonucleotide probes for target-gene sequence, be fixed on the nitrocellulose filter with horizontal wire vertical equity arrangement, preparation LipA test strip, with PCR product hybridization with biotin labeled Mycobacterium primer amplification, combine with the streptavidin of alkali phosphatase enzyme mark again, colour developing is according to judging mycobacteria strain or compound group with correspondent probe hybridization lines.
It is easy, quick that this test kit has method, 3 hours～advantage of reporting the result in 4 hours, but be only applicable to the mycobacterium culture is identified, and since the use that has at a certain bacterial classification 2～3 probes, therefore 14 probes only can be to mycobacterium tuberculosis complex, mycobacterium kansasii, mycobacterium xenopi, mycobacterium gordonae, mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum and Mycobacterium chelonei, and totally 8 kinds of mycobacteriums identify that the mycobacterium beyond the .8 kind needs to identify to planting by additive method.
Mycobacteria strain is identified in NTM disease and aspects such as diagnosis lungy, differential diagnosis, treatment and epidemiology survey significant:
1, diagnosis: smear acid-fast stain microscopy and mycobacterium separation and Culture are the methods of mainly making a definite diagnosis lungy, but because methodological restriction, even smear is difficult to also determine that it is MTB or NTM or/and cultivate the positive, therefore, need carry out mycobacteria strain and identify, make diagnosis lungy more be tending towards science.
2, differential diagnosis: NTM is sick very similar to tuberculosis clinical symptom and conducting lung X-ray examination performance, is difficult to differentiate, must rely on the strain identification result, just can clarify a diagnosis.
3, treatment: NTM is different to drug susceptibility with MTB, and most NTM are to the antitubercular agent natural drug resistance, and different N TM bacterial classification is also different to drug susceptibility, and the NTM disease should be different to some extent with the tuberculotherapy scheme.
4, epidemiology: important value is arranged in epidemiology survey.
The target gene of studying both at home and abroad at present and using more mycobacterium to detect, identify mainly is insertion sequence (insertion sequence, IS), the protein coding gene sequence, 16S rRNA gene (16S rDNA) sequence and 16S-23S rRNA gene (16S-23S rDNA) internal transcribed spacer district (internal transcribed spacer, ITS) sequence.These gene orders are broadly divided into two classes: a class is the specific sequence that exists only in mycobacterium tuberculosis complex bacterial classification high conservative, as IS6110, IS1081, protein coding gene MTP40, MPB70 and MPB64, can select to be used for the detection and the evaluation of mycobacterium tuberculosis complex, wherein IS6110 detects, identifies the gene order of normal employing of mycobacterium tuberculosis complex test kit.Another kind of be the total not only high conservative of Mycobacterium bacterial classification but also have kind between the sequence of polymorphism, the protein coding gene sequence (65KD albumen, 32KD albumen, dnaj albumen and RNA polymerase β subunit encoding gene) total as the Mycobacterium bacterial classification, 16S rDNA and 16S-23S rDNA ITS sequence can select to be used for the detection and the evaluation of mycobacteria strain.
16S rDNA is traditional target-gene sequence that division bacteria is identified on gene level.Now finished the whole or part nucleotide sequencing of kind of mycobacteria strain 16S rDNA surplus in the of 50, find by analyzing relatively, they had both contained the Mycobacterium specific nucleotide sequence, contain the special variable region on kind of the level again, mycobacterium 16S rDNA contains two variable regions, both has been positioned at the A variable region and the B variable region that is positioned at 430～500 Nucleotide of 126～267 Nucleotide.Variation is higher than the B variable region between A variable region nucleotide kind, and mycobacteria strain identifies how to carry out on the level of A variable region.But the pathogenic NTM that some is important clinically, between mycobacterium kansasii, stomach mycobacterium, Mycobacterium scrofulaceum and ape and monkey mycobacterium, Soviet Union adds between branch bacillus and the Ma Ermo mycobacterium, between Mycobacterium marinum and the mycobacterium buruli, between Mycobacterium chelonei and the mycobacterium abscessus on this level sequence identical, it can only be identified to compound group's level, can not identify to kind.
In recent years, 16S-23S rDNA ITS sequence is adopted by people as a kind of new target-gene sequence that division bacteria is identified on gene level.To kind of mycobacterium 16S-23S rDNA ITS sequencing result analysis revealed surplus 50, it is bigger that mycobacterium 16S-23S rDNA ITS sequence puts in order according to different its bases of bacterial classification and length all makes a variation, and nucleotide sequence length is from bp more than 200～bp more than 400.Include the kind distinguished sequence of 2 Mycobacterium conservative fragments and several alterable heights at whole nucleotide sequence, polymorphism is compared with 16S rDNA apparently higher than 16S rDNA between its kind, is more suitable for the evaluation of bacterial classification.Sequence is in full accord except that between Mycobacterium marinum and mycobacterium buruli can not differentiate, mycobacterium kansasii, stomach mycobacterium, Mycobacterium scrofulaceum, ape and monkey mycobacterium, Soviet Union can be added branch bacillus, Ma Ermo mycobacterium, Mycobacterium chelonei and mycobacterium abscessus and identify to planting.
Summary of the invention
The purpose of this invention is to provide a kind of easy, quick, accurately detect and the method and the dedicated kit thereof of identification of mycobacterium bacterial classification.
Mycobacteria strain provided by the present invention detects and identification kit, comprises Mycobacterium special primer and mycobacteria strain detection chip;
Described mycobacteria strain detection chip comprises the mycobacteria strain detection probes; Described mycobacteria strain detection probes is by the Mycobacterium specific probe, and mycobacterium tuberculosis complex specific probe and following mycobacterium kind specific probe are formed: mycobacterium kansasii, sea-mycobacterium buruli, the ape and monkey mycobacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, Soviet Union adds the branch bacillus, mycobacterium avium, Mycobacterium intracellulare, toad-Amur mycobacterium, the Ma Ermo mycobacterium, the soil mycobacterium, achromatic mycobacterium, the stomach mycobacterium, mycobacterium triviale, the Asia mycobacterium, the Amur mycobacterium, mycobacterium fortuitum, Mycobacterium chelonei, mycobacterium abscessus, M. smegmatics, Mycobacterium phlei, yellowish mycobacterium, pale yellow mycobacterium, the Di Shi mycobacterium, the cow mycobacterium, gold-Di Shi-Chu Bu mycobacterium, new golden mycobacterium specific probe;
Described Mycobacterium special primer is two oligonucleotide with nucleotide sequence of sequence 1 and sequence 2 in the sequence table;
Described Mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 3 in the sequence table at least;
Described mycobacterium tuberculosis complex specific probe is the oligonucleotide that has the nucleotide sequence of sequence 5 in the sequence table and/or sequence 7 at least;
Described mycobacterium kansasii specific probe is the oligonucleotide that has the nucleotide sequence of sequence 9 in the sequence table and/or sequence 10 at least;
Described sea-mycobacterium buruli specific probe is the oligonucleotide that has the nucleotide sequence of sequence 12 in the sequence table at least;
Described ape and monkey mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 14 in the sequence table and/or sequence 16 at least;
Described Mycobacterium scrofulaceum specific probe is the oligonucleotide that has the nucleotide sequence of sequence 18 in the sequence table at least;
Described mycobacterium gordonae specific probe is the oligonucleotide that has the nucleotide sequence of sequence 20 in the sequence table at least;
It is the oligonucleotide that has the nucleotide sequence of sequence 22 in the sequence table at least that described Soviet Union adds branch bacillus specific probe;
Described mycobacterium avium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 24 in the sequence table at least;
Described Mycobacterium intracellulare specific probe is the oligonucleotide that has the nucleotide sequence of sequence 26 in the sequence table at least;
Described toad-Amur mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 28 in the sequence table and/or sequence 30 at least;
Described Ma Ermo mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 32 in the sequence table at least;
Described soil mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 34 in the sequence table at least;
Described achromatic mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 36 in the sequence table at least;
Described stomach mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 38 in the sequence table at least;
Described mycobacterium triviale specific probe is the oligonucleotide that has the nucleotide sequence of sequence 40 in the sequence table at least;
Described Asia mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 42 in the sequence table at least;
Described Amur mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 44 in the sequence table at least;
Described mycobacterium fortuitum specific probe is the oligonucleotide that has the nucleotide sequence of sequence 46 in the sequence table at least;
Described Mycobacterium chelonei specific probe is the oligonucleotide that has the nucleotide sequence of sequence 48 in the sequence table and/or sequence 50 at least;
Described mycobacterium abscessus specific probe is the oligonucleotide that has the nucleotide sequence of sequence 52 in the sequence table at least;
Described M. smegmatics specific probe is the oligonucleotide that has the nucleotide sequence of sequence 54 in the sequence table at least;
Described Mycobacterium phlei specific probe is the oligonucleotide that has the nucleotide sequence of sequence 56 in the sequence table at least;
Described yellowish mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 58 in the sequence table at least;
Described pale yellow mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 60 in the sequence table at least;
Described Di Shi mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 62 in the sequence table at least;
Described cow mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 64 in the sequence table at least;
Described gold-Di Shi-Chu Bu mycobacterium specific probe is the oligonucleotide that has the nucleotide sequence of sequence 66 in the sequence table at least;
Described new golden mycobacterium is the oligonucleotide that has the nucleotide sequence of sequence 68 in the sequence table and/or sequence 70 and/or sequence 72 at least.
Above-mentioned Mycobacterium special primer, mycobacteria strain detection chip, mycobacteria strain detection probes all belong to protection scope of the present invention.
The method of utilizing the mentioned reagent box to detect mycobacterium provided by the present invention, be that genomic dna with mycobacterium sample to be measured is a template, utilize described Mycobacterium special primer to carry out pcr amplification, the pcr amplification product and the described mycobacteria strain detection chip that obtain are hybridized, determine the kind of mycobacterium to be measured according to hybridization signal.
Described mycobacterium sample to be measured is smear acid-fast stain male mycobacterium culture or smear acid-fast stain male sputum specimen.
Described hybridization is water conservancy diversion hybridization.
The water conservancy diversion hybridization technique makes traditional making nucleic acid molecular hybridization method two dimensional surface molecular interaction, rise to the three-dimensional planar molecular interaction, the passive infiltration fenestra of molecule is interacted become initiatively and interact by water conservancy diversion, improved nucleic acid molecule intermolecular hybrid efficient, shortened each operation steps time, easy and simple to handle and save reagent, reduce experimentation cost.
The present invention has following characteristics:
1, She Ji 1 pair of Mycobacterium primer is tested the 51 kinds of mycobacteria strains that try that can increase after deliberation.
2, experiment sieving is after deliberation selected 27 kinds of Mycobacterium specific probe, mycobacterium tuberculosis complex specific probe and various NTM kind specific probes, the preparation detection chip.According to hybridization signal, mycobacterium is identified.This probe has been contained mycobacterium tuberculosis complex and the various pathogenic NTM bacterial classifications of clinical visible, for enlarging the evaluation scope, has also contained the saprophytism NTM bacterial classification that accidental pathogenic NTM bacterial classification and nature extensively distribute simultaneously, and number of probes is many.It is advantageous that:
(1), is used for detecting, identifying mycobacterium tuberculosis complex:, detect, identify mycobacterium tuberculosis complex according to showing Mycobacterium probe, mycobacterium tuberculosis complex probe hybridization signal.
(2), be used for detecting, identify various NTM bacterial classifications: according to showing the Mycobacterium probe, corresponding NTM kind probe hybridization signal can detect, identify mycobacterium kansasii, sea-mycobacterium buruli, the ape and monkey mycobacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, Soviet Union adds the branch bacillus, mycobacterium avium, Mycobacterium intracellulare, mycobacterium xenopi, the Ma Ermo mycobacterium, the soil mycobacterium, achromatic mycobacterium, the stomach mycobacterium, mycobacterium triviale, the Asia mycobacterium, the Amur mycobacterium, mycobacterium fortuitum, Mycobacterium chelonei, mycobacterium abscessus, M. smegmatics, Mycobacterium phlei, yellowish mycobacterium, pale yellow mycobacterium, the Di Shi mycobacterium, the cow mycobacterium, gold-Chu Bu mycobacterium, new golden mycobacterium.
(3), be used for detecting, identifying mycobacterium tuberculosis complex and the polyinfection of NTM bacterial classification:, can detect, identify mycobacterium tuberculosis complex and the polyinfection of NTM bacterial classification according to showing Mycobacterium probe, mycobacterium tuberculosis complex probe, corresponding NTM kind probe hybridization signal.
(4), be used for detecting, differentiating mycobacterium tuberculosis complex and NTM: when detecting, identifying the NTM bacterial classification of film chip probe outside containing, then show Mycobacterium probe hybridization signal, it can be accredited as NTM, but can not identify to the kind level.
3, specimen dna method for preparing template-high-temperature cracking method to be measured has been set up in test after deliberation.
(1), solid medium upper branch bacillus culture: a little adds lysate to get culture, boils, the centrifuging and taking supernatant is used for pcr amplification.
(2), liquid nutrient medium mycobacterium intracellulare culture: after getting medium centrifugal, precipitation adds that lysate boils, the centrifuging and taking supernatant is used for pcr amplification.
(3), patient's sputum specimen: the liquefaction processing sputum specimen, the washing postprecipitation adds that lysate boils, the centrifuging and taking supernatant is used for pcr amplification.
Test kit of the present invention can not only be used for the evaluation of mycobacterium culture, and can directly detect, identify mycobacterium tuberculosis complex and NTM bacterial classification from patient's sputum specimen.
4, adopt PCR-water conservancy diversion hybridization technique, after deliberation assay optimization, determine best pcr amplification and water conservancy diversion hybridization testing conditions, detect mycobacterium susceptibility and reach 10
2～10
3/ ml mycetocyte suspension, the oligonucleotide probe high specificity, only with corresponding mycobacterium hybridization, mycobacteria strain is identified the accuracy height.
5, compare with conventional film making nucleic acid molecular hybridization technology, the operation of water conservancy diversion hybridization technique is more easy, quick, crossover process 1～1.5 hour.Whole testing process is only finished from the pcr amplification to hybridization and is needed about 3.5 hours～4 hours.And saving reagent reduces experimentation cost.
Embodiment
The present invention is a target-gene sequence with 16S-23S rDNA ITS, adopts the total target-gene sequence design Mycobacterium special primer of Mycobacterium and belongs to special oligonucleotide probe, contains the gene fragment of bacterial classification region of variability with designed primer PCR amplification.Design a series of kinds or the special oligonucleotide probe of group with the bacterial classification region of variability, arrange in certain sequence and be fixed on the nylon membrane carrier, preparation detection chip (film chip).The present invention adopts a kind of novel nucleic acid molecular hybridization method, it is PCR-water conservancy diversion hybridization (flow-through hybridization) method, the hybridization testing process adopts the water conservancy diversion hybridization instrument, crossover process can be at any time regulating effect temperature as required, its ultimate principle and working method are:
1, in triumphant general water conservancy diversion hybridization instrument (HybriMax), is fit into film chip (15 punch die);
2, in detecting the hole, add 0.8ml hybridization solution effect 2～3min, water conservancy diversion, prehybridization;
3, add the biotin labeling sex change strand pcr amplification product hybridization solution 0.5ml effect 5～10min that contains to be measured, water conservancy diversion, the strand target nucleic acid molecule when the fenestra, be fixed in fenestra in corresponding oligonucleotide probe hybridization combine, form probe biotin labeling target nucleic acid molecule mixture;
4, add washing composition 0.8ml, water conservancy diversion is not hybridized binding molecule and is passed film and be washed removing, washs three times;
5, add blocker 0.5ml, water conservancy diversion adds blocker 0.5ml effect 3～5min, water conservancy diversion, blank spot on the barrier film chip again;
6, add streptavidin-alkaline phosphatase enzyme conjugates (SA-AP) 0.5ml effect 3min, water conservancy diversion when the SA-AP molecule passes through fenestra, combines with probe-biotin labeling target nucleic acid molecule mixture;
7, add washing composition 0.8ml, water conservancy diversion, binding molecule does not pass film and is washed removing, washs four times;
8, add enzyme substrates (NBT/BCIP solution) 0.5ml effect 5～10min, water conservancy diversion, substrate colour developing;
9, add washing composition 0.8ml, water conservancy diversion, color development stopping reaction, washing secondary;
10, take out the film chip, after the distilled water rinsing, seasoning.According to corresponding genus, compound group, plant specific probe hybridization signal, identification of mycobacterium.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Embodiment 1, mycobacteria strain detect and identification kit
This test kit utilizes round pcr and water conservancy diversion hybridization technique principle, the pcr amplification product and the fixing low density film chip hybridization of Mycobacterium, mycobacterium tuberculosis complex and the special oligonucleotide probe of various NTM bacterial classifications, to the mycobacterium culture, comprise that solid medium and liquid nutrient medium culture carry out mycobacteria strain and identify; Also can directly from smear acid-fast stain (+) sputum specimen, detect and the identification of mycobacterium bacterial classification.
One, test kit is formed
Mycobacteria strain of the present invention detects and identification kit is made up of following PCR reagent and water conservancy diversion hybridizing reagent, and concrete composition is as follows:
(1) PCR reagent
| PCR reagent | Specification | Quantity |
| Lysate | 850 μ l/ pipe | 2 pipes |
| 10 times of PCR mixed solutions | 3 μ l/ pipe | 24 pipes |
| Positive reference substance | 30 μ l/ pipe | 1 pipe |
| The negative control product | 30 μ l/ pipe | 1 pipe |
| The sterilization deionized water | The 1ml/ pipe | 1 pipe |
Wherein, lysate is grouped into by following one-tenth: 20mmol/L Tris-HCl (pH8.0), 0.5% (V/V) TritonX-100,0.5% (V/V) NP-40 solution.
10 times of PCR mixed solutions are grouped into by following one-tenth: 100mmol/L Tris-HCl (pH8.3), 500mmol/L KCl, 20mmol/L MgCl
2, 0.1mmol/L EGTA-Na
2, 0.2% (0.2g/100ml) BSA, 1% (V/V) TritonX-100, each 2mmol/L dATP, dUTP, dGTP, dCTP, each 2 μ mol/L primer I, primer I I, 1.5U Taq archaeal dna polymerase/10 times of PCR mixed solutions of 3 μ l, 10 times of PCR mixed solutions of 0.5U UNG enzyme/3ul.
Wherein, Mycobacterium specific primer I sequence: 5 '-ACCTCCTTTCTAAGGAGCACC-3 ' 5 ' end biotin labeling; Mycobacterium specific primer I I sequence: 5 '-GATGCTCGCAACCACTATCCA-3 '
Positive reference substance: 10 of deactivation
6/ ml Mycobacterium tuberculosis mycetocyte suspension.
Negative control product: sterilization deionized water.
Above-mentioned PCR reagent is-20 ℃ of preservations.
(2) water conservancy diversion hybridizing reagent
| The water conservancy diversion hybridizing reagent | Specification | Quantity |
| Solution 1 (hybridization solution) | The 85ml/ bottle | 2 bottles |
| Solution 2 (blocking agent) | The 30ml/ bottle | 1 bottle |
| Solution 3 (washing composition) | The 100ml/ bottle | 1 bottle |
| Solution 4 (developer) | The 20ml/ bottle | 1 bottle |
| Enzyme labelling liquid | The 15ml/ bottle | 1 bottle |
| NBT solution | 70 μ l/ pipe | 1 pipe |
| BCIP solution | 60 μ l/ pipe | 1 pipe |
| The film chip | 1 (15 person-portion)/bag | 2 bags |
Wherein, solution 1:0.3mol/L NaCl, the 0.03mol/L trisodium citrate, 0.1% (0.g/100ml) SDS transfers pH to 7.0. with NaOH
Solution 2: blocker (Surmodics company product).
Solution 3:0.1mol/L Tris-HCl (pH7.5), 0.15mol/L NaCl, 0.1% (V/V) Tween-20.
Solution 4:0.1mol/L Tris-HCl (pH9.5), 0.1mol/L NaCl, 0.05mol/L MgCl
2
Enzyme labelling liquid is grouped into by following one-tenth: 1: 3000 times of dilution of AP stablizer (Surmodics company product) streptavidin-alkaline phosphatase enzyme conjugates (streptavidin-Alkaline Phosphate Conjugate, SA-AP (Amershum company product)).
NBT solution is grouped into by following one-tenth: 75mg/ml NBT solution, the preparation of 70% dimethyl formamide solution.
BCIP solution is grouped into by following one-tenth: 50mg/ml BCIP solution, dimethyl formamide solution preparation.
This test kit comprises film chip A, film chip A ', and film chip B, film chip B ', at least a among film chip C and the film chip C ':
The mycobacteria strain detection probes of film chip A is 29 oligonucleotide probes that the probe in the table 1 is numbered 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a, 26a, 27a, 28a, 29a.
The mycobacteria strain detection probes of film chip A ' is 29 oligonucleotide probes that the probe in the table 1 is numbered 1a ', 2a ', 3a, 4a ', 5a ', 6a ', 7a ', 8a ', 9a ', 10a ', 11a ', 12a ', 13a ', 14a ', 15a ', 16a ', 17a ', 18a ', 19a ', 20a ', 21a ', 22a ', 23a ', 24a ', 25a ', 26a ', 27a ', 28a ', 29a '.
The mycobacteria strain detection probes of film chip B ' is 29 oligonucleotide probes that the probe in the table 1 is numbered 1a ', 2b ', 3b ', 4a ', 5b ', 6a ', 7a ', 8a ', 9a ', 10a ', 11b ', 12a ', 13a ', 14a ', 15a ', 16a ', 17a ', 18a ', 19a ', 20b ', 21a ', 22a ', 23a ', 24a ', 25a ', 26a ', 27a ', 28a ', 29b '.
The mycobacteria strain detection probes of film chip B is 29 oligonucleotide probes that the probe in the table 1 is numbered 1a, 2b, 3b, 4a, 5b, 6a, 7a, 8a, 9a, 10a, 11b, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20b, 21a, 22a, 23a, 24a, 25a, 26a, 27a, 28a, 29b.
The mycobacteria strain detection probes of film chip C is 29 oligonucleotide probes that the probe in the table 1 is numbered 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a, 26a, 27a, 28a, 29c.
The mycobacteria strain detection probes of film chip C ' is 29 oligonucleotide probes that the probe in the table 1 is numbered 1a ', 2b ', 3b ', 4a ', 5b ', 6a ', 7a ', 8a ', 9a ', 10a ', 11b ', 12a ', 13a ', 14a ', 15a ', 16a ', 17a ', 18a ', 19a ', 20b ', 21a ', 22a ', 23a ', 24a ', 25a ', 26a ', 27a ', 28a ', 29c '.
Table 1. mycobacteria strain detection probes
The film chip prepares according to following method:
Get the amido modified oligonucleotide probe of 5 ' end and quantitatively be dissolved in NaHCO
3(PH8.4) in the solution, getting 5～10pmol respectively arranges in order a little and crosses on the BiodyneC nylon membrane (Pall company product) in ethyl-(3-dimethyl propyl) carbodiimide hydrochloride (EDAC) activation treatment, to be dried with after the NaOH solution soaking, distilled water wash, seal up for safekeeping after the drying, 4 ℃ of preservations are standby.
Above-mentioned water conservancy diversion hybridizing reagent is 4 ℃ of preservations.
Two, be suitable for instrument: the pcr amplification instrument of various models, the medical nucleic acid molecule quick hybridization of Hybrimax instrument, electrophoresis apparatus, horizontal strip electrophoresis groove, gel stent plate and comb.
Three, sample requirement
(1) solid medium culture such as improvement Luo Shi etc.: the mycobacterium culture of smear acid-fast stain (+).
(2) the positive culture of Bactec-T960 MGIT liquid culture bottle or BacT ALERT 3D mycobacterium tuberculosis liquid culture bottle: the mycobacterium culture of smear acid-fast stain (+).
(3) smear acid-fast stain (+) sputum specimen 3～5ml as not handling the same day, puts-20 ℃ of preservations, preservation period 1 month.
Four, working method
(1), sample preparation
1, the solid medium culture is handled: get solid medium surface growth mycobacterium thing a little to the 1.5ml centrifuge tube, add lysate 50 μ l mixings, 95 ℃～100 ℃ heating 15～20min, the centrifugal 5min of 12000rpm, getting supernatant liquor is the PCR reaction template.
2, the liquid nutrient medium culture is handled: get in interior positive culture 1～1.5ml to the 1.5ml centrifuge tube of liquid culture bottle, the centrifugal 10min of 12000rpm, eliminate supernatant liquor as far as possible, precipitation adds lysate 50 μ l mixings, 95 ℃～100 ℃ heating 15～20min, the centrifugal 5min of 12000rpm, getting supernatant liquor is the PCR reaction template.
3, sputum specimen is handled
(1), look the thick sputum degree and in sputum, add equivalent to 2 times amount liquefying agent, vibration back room temperature is placed 30～60min, middle vibration makes it fully liquefaction for several times to limpid.Liquefying agent is provided for oneself, and compound method is as follows: standby behind 4%NaOH solution and 2.94% Citric Acid, the three sodium solution autoclavings.According to measuring after 4%NaOH solution and 2.94% Citric Acid, three sodium solution equivalent mix, add N-acetyl-L halfcystine final concentration 5mg/ml, use in the 24h before using.
(2), get in sputum 1ml to the 1.5ml centrifuge tube of liquefaction back, the centrifugal 10min of 12000rpm, supernatant liquor carefully inclines.
(3), add the 1ml sterile saline in the precipitation, the vibration mixing, the centrifugal 10min of 12000rpm, supernatant liquor carefully inclines.
(4), repeating step (3), centrifugal back exhausts whole supernatant liquors with suction nozzle as far as possible.
(5), add 50 μ l lysates in the precipitation, the mixing that fully vibrates, 95 ℃～100 ℃ heating 15min, the centrifugal 5min of 12000rpm, getting supernatant liquor is the PCR reaction template.
4, the positive and negative control product are handled: get positive and negative control product, wait to melt the back mixing, get 5 μ l and add to respectively in the 1.5ml centrifuge tube, add lysate 45 μ l mixings, 95 ℃～100 ℃ heating 15～20min, the centrifugal 5min of 12000rpm, getting supernatant liquor is pcr template.
(2), pcr amplification
Number is got 10 times of PCR mixed solution pipes per sample, and every pipe adds sterilization deionized water 24 μ l mixings respectively, adds dna profiling 3 μ l mixings, centrifugal slightly back pcr amplification.Each batch increase simultaneously positive control and each portion of negative control.
Amplification condition: 37 ℃ of 10min of elder generation; 94 ℃ of 5min then: 94 ℃ of 45sec again, 59 ℃ of 45sec, 72 ℃ of 45sec totally 35 circulations; Last 72 ℃ of 7min.
(3), agarose gel electrophoresis
1, reagent: provide for oneself.
(1) 50 * Tris-glacial acetic acid (TAE) electrophoretic buffer: Tris 242.38 grams, glacial acetic acid 57.1ml, 1M EDTA (PH8.0) 50ml, adding distil water dissolve, are settled to 1000ml.Use preceding 50 times of dilutions (1 * TAE electrophoretic buffer).
(2) 10mg/ml ethidium bromide (EB) aqueous solution is put 4 ℃ and is kept in Dark Place.
(3) load sample damping fluid: 0.25% tetrabromophenol sulfonphthalein, 40% aqueous sucrose solution, put 4 ℃ of preservations.
2, method
(1) gets agarose 2 grams, add 1 * TAE damping fluid 100ml, put microwave oven internal heating or water-bath and boil dissolving.Add the 10mg/ml EB aqueous solution 10 μ l (final concentration 5 μ g/ml), mixing gently.
(2) with adhesive tape electrophoresis supporting plate two ends are sealed, put comb, the comb base should be left about the 1mm of supporting plate surface, treats that gelating soln is cooled to about 60 ℃, pours the about 2～3mm of thickness into.After treating that gel solidifies fully, carefully take out comb, remove adhesive tape, put it in the electrophoresis chamber.Pour 1 * TAE electrophoretic buffer into, do not have gel 2～3mm.EB is a carcinogen, should wear disposable glove during operation.
(3) get pcr amplification product 5 μ l, behind the adding load sample damping fluid 1 μ l mixing, add in the gel sample hole.
(4) connect power supply, electrophoresis (sample well is positioned at negative pole end) under the voltage of the 5V/cm left and right sides.
(5) electrophoresis finishes the back powered-down, takes out gel, with Ultraviolet Detector or gel imaging instrument observations.Ultraviolet ray has infringement to human eye and skin, should use the protection of against ultra-violet protective goggles and glass baffle plate.
3, result
Positive and negative control product should be distinguished (+) and (-).Clinical sample (+) amplified production carries out water conservancy diversion hybridization.
(4), water conservancy diversion hybridization
Each test sample takies compartment one hole, and following reagent dosage is the single hole consumption.
1, prepares before the experiment
(1) will hybridize the detection reagent balance to room temperature.
(2) solution 1 is preheated to 49 ℃ before use.
(3) test sample is prepared: 98 ℃ of heat denatured 5min of PCR product add 15～20 μ l sex change PCR products immediately to ice bath at least 2～5min in 0.5ml solution 1.
(4) NBT/BCIP colour developing working fluid preparation: according to consumption preparation NBT/BCIP colour developing working fluid.Basis 1ml consumption: add 4.5 μ l NBT solution in the 1ml solution 4, mixing adds 3.5 μ l BCIP solution again, uses in the mixing, 24h.
2, hybridization instrument is prepared
(1) opens the power supply of hybridization instrument.
(2) waste liquid outlet at the hybridization instrument rear portion installs the waste liquid cylinder.
(3) according to switchboard indication, selected [Manual Mode] enters temperature by [Enter] key and sets the interface, behind 49 ℃ of the input temps, confirms and heats up by [Enter] key.
(4) be full of reaction chamber with distilled water, place metallic porous sheet and open water pump, behind the moisture content above the discharge porous plate, switch off the pump.
(5) with tweezers the film chip is placed on the metallic porous sheet.
(6) plastics film with corresponding hole count is placed on the film chip.
(7) on plastics film, place silica gel seal and compartment.
(8) fix clinching lid, should aim at two holes of reaction chamber afterbody in the time of fixedly and press and be pushed forward to the end.
(9) turn on pump closes pump after draining the moisture content that remains on the film.
3, hybridization
(1) under hybridization instrument is ready to condition, hybridizes in 49 ℃ (± 1 ℃).
(2) solution 1 of adding 0.8ml preheating in detecting the hole covers cover plate and hatches 2～3min, and turn on pump is discharged solution, closes pump then.
(3) add the off-the-shelf test sample of 0.5ml in detecting the hole, cover cover plate and hatch 5～10min, turn on pump is discharged solution.
(4) opening under the condition of pump, washing film 3 times, each 0.8ml with the solution 1 of preheating.After washing film for the second time, enter the temperature modification interface by (ESC) key, behind 36 ℃ of the input temps, confirm by (Enter) key.Needn't reduce to 36 ℃ and just wash film for the third time.
(5) add 0.5ml solution 2 in detecting the hole, turn on pump is discharged solution, closes pump, adds 0.5ml solution 2 again, covers cover plate and hatches 5min, and turn on pump is discharged solution, closes pump.
When (6) temperature is 36 ℃ (± 1 ℃), add 0.5ml enzyme labelling liquid in detecting the hole, cover cover plate and hatch 3～5min, turn on pump is discharged solution.
(7) opening under the condition of pump, washing film 4 times, each 0.8ml with solution 3.
(8) turn off water pump.
(9) add 0.5ml NBT/BCIP colour developing working fluid in detecting the hole, cover cover plate colour developing 5～10min, turn on pump is discharged solution.
(10) opening under the condition of pump, washing film 2 times, each 0.8ml with solution 1.
(11) turn off water pump, open clinching lid, take out compartment, take out Hybond membrane with tweezers and put in the distilled water after the rinsing, be placed on the thieving paper.
(4), interpretation of result
Visual inspection, the positive hybridization signal of bluish voilet spot is analyzed result of determination according to hybridization signal.
Five, limitation
This test kit is used for mycobacterium tuberculosis complex and 29 kinds of NTM bacterial classifications detect and evaluation.Mycobacterium tuberculosis complex and 29 kinds of NTM bacterial classification clinical very rare mycobacteriums in addition can preliminary evaluation be NTM only, and can not identify to kind.
Six, reagent specification
PCR reagent 34 person-portions/box, water conservancy diversion hybridizing reagent 30 person-portions/box.
Seven, preservation condition
PCR reagent is stored in-20 ℃, avoids repeatedly multigelation.The water conservancy diversion hybridizing reagent is stored in 4 ℃.Validity period 8 months.
Embodiment 2, utilize the test kit of embodiment 1 to detect mycobacterium
Mycobacterium and non-branch bacillus canonical reference bacterium source are in Chinese medicine microbial strains preservation administrative center (CMCC) and Chinese common micro-organisms culture presevation administrative center (CCGMC).
The strain of mycobacterium clinical isolates: the solid medium culture is respectively from Shanghai Pulmonary Hospital (Shanghai), Beijing Tuberculosis and Thoracic Tumor Research Institute (Beijing 1), ChangPing, Beijing City district Center for Disease Control (Beijing 2), Shenzhen's Center for Disease Control (Shenzhen), Guangzhou chest hospital (Guangzhou), Nanping City in Fujian province Center for Disease Control (Fujian).
The liquid nutrient medium culture is respectively from Shenyang Hospital for Thoracics (Shenyang), Chongqing City chest hospital (Chongqing) and PLA General Hospital second affiliated hospital (Beijing 3).
Sputum specimen is from hospital of Beijing's lung section (Beijing 4), Beijing 1 and Beijing 3.
Utilize the test kit of embodiment 1 (to comprise film chip A, film chip A ', film chip B, film chip B ', film chip C and film chip C ') according to the method for embodiment 1, detect 51 kinds of mycobacteriums and 10 kinds of non-branch bacillus canonical reference bacterial strains in the table 2, the mycobacterium clinical separation strain in the table 3.Film chip A, film chip A ', film chip B, film chip B ', the detected result of film chip C and film chip C ' all conforms to canonical reference bacterial strain situation, and conforms to the 16S-23S rDNA ITS order-checking qualification result of bacterial strain.
Wherein the detected result of film chip A is shown in table 2 and table 3:
Table 2 mycobacterium and non-branch bacillus canonical reference bacterial strain pcr amplification and and specific probe results of hybridization
Table 3
Mycobacterium clinical separation strain (solid medium culture) qualification result
Utilize the method for the test kit (comprising film chip A, film chip A ', film chip B, film chip B ', film chip C and film chip C ') of embodiment 1, tracer liquid culture medium culturing thing and sputum specimen according to embodiment 1.The detected result of film chip A, film chip A ', film chip B, film chip B ', film chip C and film chip C ' all conforms to the 16S-23S rDNA ITS order-checking qualification result of bacterial strain: 81 parts of liquid nutrient medium cultures (Shenyang), it is mycobacterium tuberculosis that test kit is identified 69 parts, and 12 parts is Mycobacterium intracellulare; 2 parts of liquid nutrient medium cultures (Chongqing), it is mycobacterium abscessus that test kit is identified 1 part, 1 part is mycobacterium fortuitum; 5 parts of liquid nutrient medium cultures (Beijing 3), it is Mycobacterium intracellulare that test kit is identified 1 part, 4 parts is mycobacterium abscessus; 110 parts of acid-fast stain positive spit samples (+～++ ++) (Beijing 1, Beijing 3, Beijing 4), it is mycobacterium tuberculosis that test kit is identified 102 parts, and 2 parts is Mycobacterium intracellulare, and 2 parts is Mycobacterium scrofulaceum, and 4 parts is mycobacterium abscessus.
Sequence table
<160>73
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
acctcctttc taaggagcac c 21
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
gatgctcgca accactatcc a 21
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
aaacaccaca ccccaccac 19
<210>4
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
ctcaaacacc acaccccacc accaa 25
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
tgtgttggtg gccaactttg 20
<210>6
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
gtgtgttggt ggccaacttt gttgtcatgc 30
<210>7
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
cacggcctac gccccacca 19
<210>8
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>8
acccctcacg gcctacgccc caccagttgg 30
<210>9
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
acaactctcg aacagagccc 20
<210>10
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>10
gacaactctc gaacagagc 19
<210>11
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>11
gggacaactc tcgaacagag cccgagtgtg 30
<210>12
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>12
catcccgaaa ccaacagaga 20
<210>13
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>13
acaacatccc gaaaccaaca gagatgttgc 30
<210>14
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>14
tgtctggcga ttgcctgttg 20
<210>15
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>15
gtgtctggcg attgcctgtt gttgt 25
<210>16
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>16
cttcaaccga agtcggccg 19
<210>17
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>17
acaccacttc aaccgaagtc ggccgagtgt 30
<210>18
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>18
caccactcag aacgagccga 20
<210>19
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>19
gagggacacc actcagaacg agccgagtgt 30
<210>20
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>20
ggacagcacc cgagggtg 18
<210>21
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>21
ggggacagca cccgagggtg ttgcc 25
<210>22
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>22
acaacagctc tggccaagcc 20
<210>23
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>23
tgggacaaca gctctggcca agcctgagtg 30
<210>24
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>24
cacacggacg gaccgagt 18
<210>25
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>25
actccacacg gacggaccga gtgtt 25
<210>26
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>26
cacacggatc gaccgagt 18
<210>27
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>27
actccacacg gatcgaccga gtgtt 25
<210>28
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>28
cggttactgc ctgctgcc 18
<210>29
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>29
tgccggcggt tactgcctgc tgcccaacac 30
<210>30
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>30
tcggcccgac gccacaaca 19
<210>31
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>31
aacacctcgg cccgacgcca caacacgccg 30
<210>32
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>32
ggacaccacg cggactgg 18
<210>33
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>33
gggacaccac gcggactggc gagtg 25
<210>34
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>34
gttgttcgta attcctctgc acc 23
<210>35
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>35
gcttgttgtt gttcgtaatt cctctgcacc 30
<210>36
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>36
ttgttgctcg taaattttct gcac 24
<210>37
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>37
tttgttgttg ctcgtaaatt ttctgcacca 30
<210>38
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>38
caacactctt ggacgagtcc 20
<210>39
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>39
gacaacactc ttggacgagt ccaagacaag 30
<210>40
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>40
accacttgat gcgacggttc 20
<210>41
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>41
tcaacaccac ttgatgcgac ggttcgttgt 30
<210>42
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>42
acggcctgca tcccacca 18
<210>43
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>43
ctccacacgg cctgcatccc accaa 25
<210>44
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>44
cggctcgact tccgccca 18
<210>45
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>45
cctcacggct cgacttccgc ccact 25
<210>46
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>46
gcaaccggtt cctcacgg 18
<210>47
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>47
ctacaggcaa ccggttcctc acggc 25
<210>48
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>48
caccaagcga gtaaccacta c 21
<210>49
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>49
ttcaccaagc gagtaaccac tacag 25
<210>50
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>50
actatccata agtcaaagac ca 22
<210>51
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>51
actatccata agtcaaagac cacac 25
<210>52
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>52
tcaccaagta gatatccact ac 22
<210>53
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>53
ttcaccaagt agatatccac tacag 25
<210>54
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>54
tccccaacgg agagtgtctc 20
<210>55
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>55
gctcacaccc tccccaacgg agagtgtctc 30
<210>56
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>56
ccctacctct tatagggcac 20
<210>57
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>57
ccgcgcaact ccctacctct tatagggcac 30
<210>58
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>58
ttcctcaaag cagtgttccc 20
<210>59
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>59
gatgattcct caaagcagtg ttcccacacc 30
<210>60
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>60
ctgatgactt cgcaccacaa 20
<210>61
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>61
gtctgatgac ttcgcaccac aaccccggaa 30
<210>62
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>62
cgtccgaacc agaatcgctt 20
<210>63
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>63
cgtccgaacc agaatcgctt ccgggactcg 30
<210>64
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>64
tcggccaagt ttgtcatgtg 20
<210>65
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>65
taccggctcg gccaagtttg tcatgtgcac 30
<210>66
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>66
cgaatcgcct gattcaccg 19
<210>67
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>67
ccaacgaatc gcctgattca ccgaa 25
<210>68
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>68
gaatcgacca aggtcaccga 20
<210>69
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>69
aacgaatcga ccaaggtcac cgaac 25
<210>70
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>70
ttctccgtag agcgaaggtc 20
<210>71
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>71
ccttctccgt agagcgaagg tcccg 25
<210>72
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>72
gacaacagca accgcctgac 20
<210>73
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>73
ggcgacaaca gcaaccgcct gaccc 25
Claims (3)
1. mycobacteria strain detects and identification kit, comprises Mycobacterium special primer and mycobacteria strain detection chip;
Described mycobacteria strain detection chip comprises the mycobacteria strain detection probes; Described mycobacteria strain detection probes is by the Mycobacterium specific probe, and mycobacterium tuberculosis complex specific probe and following 27 kinds of mycobacterium kind specific probes are formed: mycobacterium kansasii, sea-mycobacterium buruli, the ape and monkey mycobacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, Soviet Union adds the branch bacillus, mycobacterium avium, Mycobacterium intracellulare, toad-Amur mycobacterium, the Ma Ermo mycobacterium, the soil mycobacterium, achromatic mycobacterium, the stomach mycobacterium, mycobacterium triviale, the Asia mycobacterium, the Amur mycobacterium, mycobacterium fortuitum, Mycobacterium chelonei, mycobacterium abscessus, M. smegmatics, Mycobacterium phlei, yellowish mycobacterium, pale yellow mycobacterium, the Di Shi mycobacterium, the cow mycobacterium, gold-Di Shi-Chu Bu mycobacterium, new golden mycobacterium specific probe;
Described Mycobacterium special primer is two oligonucleotide sequences shown in sequence 1 and the sequence 2 in the sequence table;
Described Mycobacterium specific probe is sequence 3 or a sequence 4 in the sequence table;
Described mycobacterium tuberculosis complex specific probe is following 1) to 4) in any: 1) sequence 5 and/or sequence 7 in the sequence table; 2) sequence 6 and/or sequence 7 in the sequence table; 3) sequence 5 and/or sequence 8 in the sequence table; 4) sequence 6 and/or sequence 8 in the sequence table;
Described mycobacterium kansasii specific probe is following 5) to 6) in any: 5) sequence 9 and/or sequence 10 in the sequence table; 6) sequence 9 and/or sequence 11;
Described sea-mycobacterium buruli specific probe is sequence 12 or a sequence 13 in the sequence table;
Described ape and monkey mycobacterium specific probe is following 7) to 10) in any: 7) sequence 14 and/or sequence 16 in the sequence table; 8) sequence 15 and/or sequence 16 in the sequence table; 9) sequence 14 and/or sequence 17 in the sequence table; 10) sequence 15 and/or sequence 17 in the sequence table;
Described Mycobacterium scrofulaceum specific probe is sequence 18 or a sequence 19 in the sequence table;
Described mycobacterium gordonae specific probe is sequence 20 or a sequence 21 in the sequence table;
It is sequence 22 or sequence 23 in the sequence table that described Soviet Union adds branch bacillus specific probe;
Described mycobacterium avium specific probe is sequence 24 or a sequence 25 in the sequence table;
Described Mycobacterium intracellulare specific probe is sequence 26 or a sequence 27 in the sequence table;
Described toad-Amur mycobacterium specific probe is following 11) to 14) in any: 11) sequence 28 and/or sequence 30 in the sequence table; 12) sequence 29 and/or sequence 30 in the sequence table; 13) sequence 28 and/or sequence 31 in the sequence table; 14) sequence 29 and/or sequence 31 in the sequence table;
Described Ma Ermo mycobacterium specific probe is sequence 32 or a sequence 33 in the sequence table;
Described soil mycobacterium specific probe is sequence 34 or a sequence 35 in the sequence table;
Described achromatic mycobacterium specific probe is sequence 36 or a sequence 37 in the sequence table;
Described stomach mycobacterium specific probe is sequence 38 or a sequence 39 in the sequence table;
Described mycobacterium triviale specific probe is sequence 40 or a sequence 41 in the sequence table;
Described Asia mycobacterium specific probe is sequence 42 or a sequence 43 in the sequence table;
Described Amur mycobacterium specific probe is sequence 44 or a sequence 45 in the sequence table;
Described mycobacterium fortuitum specific probe is sequence 46 or a sequence 47 in the sequence table;
Described Mycobacterium chelonei specific probe is following 15) to 18) in any: 15) sequence 48 and/or sequence 50 in the sequence table; 16) sequence 48 and/or sequence 51 in the sequence table; 17) sequence 49 and/or sequence 50 in the sequence table; 18) sequence 49 and/or sequence 51 in the sequence table;
Described mycobacterium abscessus specific probe is sequence 52 or a sequence 53 in the sequence table;
Described M. smegmatics specific probe is sequence 54 or a sequence 55 in the sequence table;
Described Mycobacterium phlei specific probe is sequence 56 or a sequence 57 in the sequence table;
Described yellowish mycobacterium specific probe is sequence 58 or a sequence 59 in the sequence table;
Described pale yellow mycobacterium specific probe is sequence 60 or a sequence 61 in the sequence table;
Described Di Shi mycobacterium specific probe is sequence 62 or a sequence 63 in the sequence table;
Described cow mycobacterium specific probe is sequence 64 or a sequence 65 in the sequence table;
Described gold-Di Shi-Chu Bu mycobacterium specific probe is sequence 66 or a sequence 67 in the sequence table;
Described new golden mycobacterium is following 19) to 22) in any: 19) sequence 68 and/or sequence 70 and/or sequence 72 in the sequence table; 20) sequence 69 and/or sequence 70 and/or sequence 72 in the sequence table; 21) sequence 68 and/or sequence 71 and/or sequence 73 in the sequence table; 22) sequence 69 and/or sequence 71 and/or sequence 73 in the sequence table.
2. mycobacteria strain detection chip, comprise the Mycobacterium specific probe, mycobacterium tuberculosis complex specific probe and following mycobacterium kind specific probe: mycobacterium kansasii, sea-mycobacterium buruli, the ape and monkey mycobacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, Soviet Union adds the branch bacillus, mycobacterium avium, Mycobacterium intracellulare, toad-Amur mycobacterium, the Ma Ermo mycobacterium, the soil mycobacterium, achromatic mycobacterium, the stomach mycobacterium, mycobacterium triviale, the Asia mycobacterium, the Amur mycobacterium, mycobacterium fortuitum, Mycobacterium chelonei, mycobacterium abscessus, M. smegmatics, Mycobacterium phlei, yellowish mycobacterium, pale yellow mycobacterium, the Di Shi mycobacterium, the cow mycobacterium, gold-Di Shi-Chu Bu mycobacterium, new golden branch bacillus specie specific probe;
Described Mycobacterium specific probe is sequence 3 or a sequence 4 in the sequence table;
Described mycobacterium tuberculosis complex specific probe is following 1) to 4) in any: 1) sequence 5 and/or sequence 7 in the sequence table; 2) sequence 6 and/or sequence 7 in the sequence table; 3) sequence 5 and/or sequence 8 in the sequence table; 4) sequence 6 and/or sequence 8 in the sequence table;
Described mycobacterium kansasii specific probe is following 5) to 6) in any: 5) sequence 9 and/or sequence 10 in the sequence table; 6) sequence 9 and/or sequence 11;
Described sea-mycobacterium buruli specific probe is sequence 12 or a sequence 13 in the sequence table;
Described ape and monkey mycobacterium specific probe is following 7) to 10) in any: 7) sequence 14 and/or sequence 16 in the sequence table; 8) sequence 15 and/or sequence 16 in the sequence table; 9) sequence 14 and/or sequence 17 in the sequence table; 10) sequence 15 and/or sequence 17 in the sequence table;
Described Mycobacterium scrofulaceum specific probe is sequence 18 or a sequence 19 in the sequence table;
Described mycobacterium gordonae specific probe is sequence 20 or a sequence 21 in the sequence table;
It is sequence 22 or sequence 23 in the sequence table that described Soviet Union adds branch bacillus specific probe;
Described mycobacterium avium specific probe is sequence 24 or a sequence 25 in the sequence table;
Described Mycobacterium intracellulare specific probe is sequence 26 or a sequence 27 in the sequence table;
Described toad-Amur mycobacterium specific probe is following 11) to 14) in any: 11) sequence 28 and/or sequence 30 in the sequence table; 12) sequence 29 and/or sequence 30 in the sequence table; 13) sequence 28 and/or sequence 31 in the sequence table; 14) sequence 29 and/or sequence 31 in the sequence table;
Described Ma Ermo mycobacterium specific probe is sequence 32 or a sequence 33 in the sequence table;
Described soil mycobacterium specific probe is sequence 34 or a sequence 35 in the sequence table;
Described achromatic mycobacterium specific probe is sequence 36 or a sequence 37 in the sequence table;
Described stomach mycobacterium specific probe is sequence 38 or a sequence 39 in the sequence table;
Described mycobacterium triviale specific probe is sequence 40 or a sequence 41 in the sequence table;
Described Asia mycobacterium specific probe is sequence 42 or a sequence 43 in the sequence table;
Described Amur mycobacterium specific probe is sequence 44 or a sequence 45 in the sequence table;
Described mycobacterium fortuitum specific probe is sequence 46 or a sequence 47 in the sequence table;
Described Mycobacterium chelonei specific probe is following 15) to 18) in any: 15) sequence 48 and/or sequence 50 in the sequence table; 16) sequence 48 and/or sequence 51 in the sequence table; 17) sequence 49 and/or sequence 50 in the sequence table; 18) sequence 49 and/or sequence 51 in the sequence table;
Described mycobacterium abscessus specific probe is sequence 52 or a sequence 53 in the sequence table;
Described M. smegmatics specific probe is sequence 54 or a sequence 55 in the sequence table;
Described Mycobacterium phlei specific probe is sequence 56 or a sequence 57 in the sequence table;
Described yellowish mycobacterium specific probe is sequence 58 or a sequence 59 in the sequence table;
Described pale yellow mycobacterium specific probe is sequence 60 or a sequence 61 in the sequence table;
Described Di Shi mycobacterium specific probe is sequence 62 or a sequence 63 in the sequence table;
Described cow mycobacterium specific probe is sequence 64 or a sequence 65 in the sequence table;
Described gold-Di Shi-Chu Bu mycobacterium specific probe is sequence 66 or a sequence 67 in the sequence table;
Described new golden mycobacterium is following 19) to 22) in any: 19) sequence 68 and/or sequence 70 and/or sequence 72 in the sequence table; 20) sequence 69 and/or sequence 70 and/or sequence 72 in the sequence table; 21) sequence 68 and/or sequence 71 and/or sequence 73 in the sequence table; 22) sequence 69 and/or sequence 71 and/or sequence 73 in the sequence table.
3. be used for the probe that mycobacteria strain detects, by the Mycobacterium specific probe, mycobacterium tuberculosis complex specific probe and mycobacterium kind specific probe are formed; Described mycobacterium kind specific probe is made up of following probe: mycobacterium kansasii, sea-mycobacterium buruli, the ape and monkey mycobacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, Soviet Union adds the branch bacillus, mycobacterium avium, Mycobacterium intracellulare, toad-Amur mycobacterium, the Ma Ermo mycobacterium, the soil mycobacterium, achromatic mycobacterium, the stomach mycobacterium, mycobacterium triviale, the Asia mycobacterium, the Amur mycobacterium, mycobacterium fortuitum, Mycobacterium chelonei, mycobacterium abscessus, M. smegmatics, Mycobacterium phlei, yellowish mycobacterium, pale yellow mycobacterium, the Di Shi mycobacterium, the cow mycobacterium, gold-Di Shi-Chu Bu mycobacterium, new golden branch bacillus specie specific probe;
Described Mycobacterium specific probe is sequence 3 or a sequence 4 in the sequence table;
Described mycobacterium tuberculosis complex specific probe is following 1) to 4) in any: 1) sequence 5 and/or sequence 7 in the sequence table; 2) sequence 6 and/or sequence 7 in the sequence table; 3) sequence 5 and/or sequence 8 in the sequence table; 4) sequence 6 and/or sequence 8 in the sequence table;
Described mycobacterium kansasii specific probe is following 5) to 6) in any: 5) sequence 9 and/or sequence 10 in the sequence table; 6) sequence 9 and/or sequence 11;
Described sea-mycobacterium buruli specific probe is sequence 12 or a sequence 13 in the sequence table;
Described ape and monkey mycobacterium specific probe is following 7) to 10) in any: 7) sequence 14 and/or sequence 16 in the sequence table; 8) sequence 15 and/or sequence 16 in the sequence table; 9) sequence 14 and/or sequence 17 in the sequence table; 10) sequence 15 and/or sequence 17 in the sequence table;
Described Mycobacterium scrofulaceum specific probe is sequence 18 or a sequence 19 in the sequence table;
Described mycobacterium gordonae specific probe is sequence 20 or a sequence 21 in the sequence table;
It is sequence 22 or sequence 23 in the sequence table that described Soviet Union adds branch bacillus specific probe;
Described mycobacterium avium specific probe is sequence 24 or a sequence 25 in the sequence table;
Described Mycobacterium intracellulare specific probe is sequence 26 or a sequence 27 in the sequence table;
Described toad-Amur mycobacterium specific probe is following 11) to 14) in any: 11) sequence 28 and/or sequence 30 in the sequence table; 12) sequence 29 and/or sequence 30 in the sequence table; 13) sequence 28 and/or sequence 31 in the sequence table; 14) sequence 29 and/or sequence 31 in the sequence table;
Described Ma Ermo mycobacterium specific probe is sequence 32 or a sequence 33 in the sequence table;
Described soil mycobacterium specific probe is sequence 34 or a sequence 35 in the sequence table;
Described achromatic mycobacterium specific probe is sequence 36 or a sequence 37 in the sequence table;
Described stomach mycobacterium specific probe is sequence 38 or a sequence 39 in the sequence table;
Described mycobacterium triviale specific probe is sequence 40 or a sequence 41 in the sequence table;
Described Asia mycobacterium specific probe is sequence 42 or a sequence 43 in the sequence table;
Described Amur mycobacterium specific probe is sequence 44 or a sequence 45 in the sequence table;
Described mycobacterium fortuitum specific probe is sequence 46 or a sequence 47 in the sequence table;
Described Mycobacterium chelonei specific probe is following 15) to 18) in any: 15) sequence 48 and/or sequence 50 in the sequence table; 16) sequence 48 and/or sequence 51 in the sequence table; 17) sequence 49 and/or sequence 50 in the sequence table; 18) sequence 49 and/or sequence 51 in the sequence table;
Described mycobacterium abscessus specific probe is sequence 52 or a sequence 53 in the sequence table;
Described M. smegmatics specific probe is sequence 54 or a sequence 55 in the sequence table;
Described Mycobacterium phlei specific probe is sequence 56 or a sequence 57 in the sequence table;
Described yellowish mycobacterium specific probe is sequence 58 or a sequence 59 in the sequence table;
Described pale yellow mycobacterium specific probe is sequence 60 or a sequence 61 in the sequence table;
Described Di Shi mycobacterium specific probe is sequence 62 or a sequence 63 in the sequence table;
Described cow mycobacterium specific probe is sequence 64 or a sequence 65 in the sequence table;
Described gold-Di Shi-Chu Bu mycobacterium specific probe is sequence 66 or a sequence 67 in the sequence table;
Described new golden mycobacterium is following 19) to 22) in any: 19) sequence 68 and/or sequence 70 and/or sequence 72 in the sequence table; 20) sequence 69 and/or sequence 70 and/or sequence 72 in the sequence table; 21) sequence 68 and/or sequence 71 and/or sequence 73 in the sequence table; 22) sequence 69 and/or sequence 71 and/or sequence 73 in the sequence table.
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| CN200610000496A CN1995380B (en) | 2006-01-05 | 2006-01-05 | Method for detecting and identifying mycobacterium tuberculosis strain and its dedicated reagent kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2677040A1 (en) * | 2012-06-22 | 2013-12-25 | Taichung Veterans General Hospital | Probe, chip, kit and method for detection of mycobacterium tuberculosis, non-tuberculous mycobacteria and drug resistant of mycobacterium tuberculosis |
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| US7998708B2 (en) * | 2006-03-24 | 2011-08-16 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
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| CN102229999A (en) * | 2011-06-01 | 2011-11-02 | 亚能生物技术（深圳）有限公司 | Kit for identifying Mycobacterium tuberculosis and nontuberculous mycobacteria and application method thereof |
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| CN109628619B (en) * | 2019-01-03 | 2022-05-03 | 首都医科大学附属北京胸科医院 | SNP molecular marker and method for identifying mycobacteria, primer composition, kit and application |
| CN110438205B (en) * | 2019-07-31 | 2023-07-07 | 天津市泌尿外科研究所 | Kit for detecting pathogenic mycobacterium tuberculosis by combining multiplex PCR (polymerase chain reaction) nest type PCR and touchdown PCR |
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| CN1444662A (en) * | 2000-05-30 | 2003-09-24 | 百奥麦雷公司 | Diagnosis kit for mycobacterium species identification and drug-resistance detection and mfg. method thereof |
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